CN104387454A - Method for preparing triptorelin by using fragment condensation - Google Patents
Method for preparing triptorelin by using fragment condensation Download PDFInfo
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- CN104387454A CN104387454A CN201410743707.1A CN201410743707A CN104387454A CN 104387454 A CN104387454 A CN 104387454A CN 201410743707 A CN201410743707 A CN 201410743707A CN 104387454 A CN104387454 A CN 104387454A
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Abstract
The invention discloses a method for preparing triptorelin by using fragment condensation. The method comprises the following steps of preparing a fully-protected first peptide fragment sequence of triptorelin by a solid-phase method; preparing a fully-protected second peptide fragment sequence of triptorelin by a liquid-phase method and removing amino-terminal protective groups; in the liquid phase, condensing the amino-terminal protecting group-removed fully-protected second peptide fragment sequence and the first peptide fragment sequence to obtain fully-protected triptorelin; removing side-chain protective groups of fully-protected triptorelin to obtain triptorelin crude peptide, purifying and carrying out exchange salt by virtue of high performance liquid chromatography to obtain triptorelin acetate, wherein the first peptide fragment sequence is the first-seventh amino acids of the sequence of triptorelin and the second peptide fragment sequence is the eighth-tenth amino acids of the sequence of triptorelin. By the solid-phase method and liquid-phase method in combination with fragment condensation, triptorelin is prepared, the yield and purity are increased, the cost is low and the method is conducive to mass production.
Description
Technical field
The present invention relates to pharmacy field, be specifically related to a kind of method that fragment condensation prepares triptorelin.
Background technology
Triptorelin, English name: Triptorelin, trade(brand)name: Diphereline, Wy-42462, peptide sequence is: H-Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH
2, molecular formula: C
64h
82n
18o
13, molecular weight: 1310.63.
Triptorelin, as a kind of gonadotropin releasing hormone analogues, mainly acts on prepituitary gland, is clinically used for the treatment of dependency hormonal conditions, comprises advanced prostate cancer, endometriosis, hysteromyoma, in vitro fertilization, Central precocious puberty disease etc.
The preparation method of current triptorelin is mainly based on condensation one by one.Such as patent US4010125 uses Benzhydryl amine resin to be starting raw material; with the amino acid of Boc protection for monomer; patent CN200710044419.7 and CN201310013712.2 and CN201310014882.2; all with Rink Amide mbha resin or RinkAmide AM resin for starting raw material; with the amino acid of Fmoc protection for monomer, connect amino acid one by one successively, cracking obtains triptorelin crude product; last HPLC carries out separation and purification, obtains target product.
Above method has the following problem: a) there is Arg-Pro in triptorelin sequence, need excessive amino acid under normal conditions, and condensation efficiency is low, easily occurs defect peptide with solid phase method condensation.This be cause due to amino acid self structure sterically hinderedly make condensation reaction very difficult.B) use Rink resin carboxy terminal amino acid substitution value can not be too high, synthesizes uneconomical; And it is expensive relative to the chloro-trityl chloride resin of 2-.C), during full liquid phase synthesis, good post-processing technology is needed.
So those skilled in the art still expect to obtain the product with better quality with high yield, low cost.
Summary of the invention
The present invention is directed to existing preparation method's yield low, and purifying products difficulty, be difficult to obtain the problem of highly purified triptorelin, provide the method for a kind of solid liquid phase binding fragment condensation to prepare triptorelin, improve productive rate and purity; Owing to adopting the chloro-trityl chloride resin of 2-to make cost reduce, be beneficial to scale operation.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
Scheme one:
Fragment condensation prepares a method for triptorelin, and solid phase prepares the first peptide fragment sequences of the full guard of triptorelin; Liquid phase is prepared the second peptide fragment sequences of the full guard of triptorelin and is removed its aminoterminal protecting group; In liquid phase, be stripped of the second peptide fragment sequences and the first peptide fragment sequences condensation of the full guard of aminoterminal protecting group, obtain the triptorelin of full guard; The Side chain protective group sloughing the triptorelin of full guard obtains the thick peptide of triptorelin, and high performance liquid phase purifying changes salt and obtains triptorelin acetate;
Wherein,
The first described peptide fragment sequences is 1st ~ 7 amino acids in triptorelin sequence,
The second described peptide fragment sequences is 8th ~ 10 amino acids in triptorelin sequence.
The method that above-mentioned fragment condensation prepares triptorelin preferably includes following steps:
(1) solid phase prepares the first peptide fragment sequences resin of the full guard of triptorelin,
Described full guard first peptide fragment sequences is:
Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp(Boc)-Leu-OH，
By full guard first peptide fragment sequences from cracking solid phase carrier;
(2) the second peptide fragment sequences of the full guard of liquid phase synthesis triptorelin,
Second peptide fragment sequences of described full guard is:
Fmoc-Arg(Pbf)-Pro-Gly-NH
2；
Remove the aminoterminal protecting group of the second peptide fragment sequences of full guard;
(3) the first peptide fragment sequences condensation of the full guard that the second peptide fragment sequences being stripped of the full guard of aminoterminal protecting group step (2) obtained in liquid phase and step (1) obtain obtains full guard triptorelin;
(4) Side chain protective group is sloughed in the cracking of full guard triptorelin and obtain triptorelin crude product;
(5) the purified salt that turns of triptorelin crude product obtains triptorelin acetate.
In step (1), the first peptide fragment sequences resin of full guard is coupled at successively on solid phase carrier by amino acid to obtain; Wherein, solid phase carrier used is acid sensitive resin, is preferably the chloro-trityl chloride resin of 2-.
Solid phase is prepared in the process of triptorelin full guard first peptide fragment sequences resin, and the amino deprotecting regent used is the DMF solution of the DBU of 1% for volumn concentration is the DMF solution of the piperidines of 20% or volumn concentration; Preferred volume percentage composition is the DMF solution of 20% piperidines.
Coupling agent used is HBTU and HOBt and the combination of the DIEA combination of 1:1:2 in molar ratio or DIC and the HOBt combination of 1:1 in molar ratio or PyBOP and HOBt and DIEA 1:1:2 in molar ratio.The combination of preferred HBTU and HOBt and DIEA 1:1:2 in molar ratio; Treat that the amino acid of coupling and the mol ratio of HOBt are 1:1.
The cracking agent used for volumn concentration be the DCM solution of the TFA of 0.5 ~ 1%, volumn concentration is the DCM solution of the TFE of 20% or TFE and AcOH and the DCM mixture according to volume ratio 1:2:7.Preferred volume percentage composition is the DCM solution of 0.5 ~ 1%TFA.
In step (2), the method for the second peptide fragment sequences of liquid phase synthesis full guard is, first Fmoc-Pro-OH and H-Gly-NH
2be obtained by reacting Fmoc-Pro-Gly-NH
2, slough Fmoc protection after and Fmoc-Arg (Pbf)-OH be obtained by reacting Fmoc-Arg (Pbf)-Pro-Gly-NH
2.
The described aminoterminal protecting group removing the second peptide fragment sequences of full guard namely sloughs Fmoc-Arg (Pbf)-Pro-Gly-NH
2fmoc protection, obtain H-Arg (Pbf)-Pro-Gly-NH
2.
Amino deprotecting regent is the DMF solution of the piperidines of volumn concentration 16%, or volumn concentration is the DMF solution of the DBU of 1%, or volumn concentration is the DCM solution of the diethylamine of 50%; Preferred volume percentage composition is the DCM solution of 50% diethylamine.The condensing agent that condensation reaction uses is the combination of HBTU and HOBt and the DIEA combination of 1:1:2 in molar ratio or HBTU and HOAt and the DIEA combination of 1:1:2 in molar ratio or DIC and the HOBt combination of 1:1 in molar ratio or EDC and the HOBt combination of 1:1 in molar ratio or PyBOP and HOBt and DIEA 1:1:2 in molar ratio, the combination of preferred DIC and HOBt 1:1 in molar ratio; The solvent of condensation reaction is any one or a few the combination in THF, DMF, DCM, NMP and DMSO, preferred THF.
In step (3), first peptide fragment sequences condensation of the full guard that the second peptide fragment sequences being stripped of the full guard of aminoterminal protecting group step (2) obtained and step (1) obtain obtains in full guard triptorelin process, the condensing agent used is the combination of HBTU and HOBt and DIEA 1:1:2 in molar ratio, or the combination of HBTU and HOAt and DIEA 1:1:2 in molar ratio, or the combination of EDC and HOBt 1:1 in molar ratio, or the combination of DIC and HOBt 1:1 in molar ratio, or the combination of PyBOP and HOBt and DIEA 1:1:2 in molar ratio, the combination of preferred HBTU and HOBt and DIEA 1:1:2 in molar ratio, the solvent of condensation reaction is any one or a few the combination in THF, DMF, DCM, NMP and DMSO, preferred DMF.
In step (4), the lysate that the cracking of full guard triptorelin uses is TFA and H
2o is the mixing solutions of 95:5 or TFA and EDT and TIS and PhOH and H by volume
2o is the mixing solutions of 80:5:5:5:5 or TFA and EDT and TIS and H by volume
2the mixing solutions of O 92.5:2.5:2.5:2.5 by volume.Preferred TFA and EDT and TIS and H
2the mixing solutions of O 92.5:2.5:2.5:2.5 by volume.
In step (5), purifying is that RPLC purifying changes salt, and namely chromatographic column is C18 post; Moving phase is 0.25%v/v aqueous acetic acid and 80%v/v acetonitrile solution.
Scheme two:
Fragment condensation prepares a method for triptorelin, and solid phase prepares the tripeptide fragment sequence resin of the full guard of triptorelin, then the tripeptide fragment sequence of full guard is got off from cracking resin, with H-Gly-NH in liquid phase
2condensation obtains the triptorelin of full guard, and then slough side chain protected and obtain triptorelin crude product, purifying turns salt and obtains triptorelin acetate;
Wherein, described tripeptide fragment sequence is the 1-9 amino acids in triptorelin sequence.
Above-mentioned fragment condensation prepares the method for triptorelin, preferably includes following steps:
A () solid phase prepares the tripeptide fragment sequence resin of the full guard of triptorelin,
The tripeptide fragment sequence of the full guard of described triptorelin is:
Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp(Boc)-Leu-Arg(Pbf)-Pro-OH，
By the tripeptide fragment sequence of full guard from cracking solid phase carrier;
Full guard tripeptide fragment sequence and H-Gly-NH in (b) liquid phase
2condensation obtains the triptorelin of full guard;
C the triptorelin cracking of full guard is sloughed Side chain protective group and is obtained triptorelin crude product by ();
D the purified salt that turns of () triptorelin crude product obtains triptorelin acetate.
In step (a), the tripeptide fragment sequence resin of the full guard of triptorelin is coupled at successively on solid phase carrier by amino acid to obtain peptide resin; Wherein, solid phase carrier used is acid sensitive resin, is preferably the chloro-trityl chloride resin of 2-.
Solid phase is prepared in the process of tripeptide fragment sequence resin of the full guard of triptorelin, and the amino deprotecting regent used is the DMF solution of the DBU of 1% for volumn concentration is the DMF solution of the piperidines of 20% or volumn concentration; Preferred volume percentage composition is the DMF solution of 20% piperidines.
Coupling agent used is HBTU and HOBt and the combination of the DIEA combination of 1:1:2 in molar ratio or DIC and the HOBt combination of 1:1 in molar ratio or PyBOP and HOBt and DIEA 1:1:2 in molar ratio.The combination of preferred HBTU and HOBt and DIEA 1:1:2 in molar ratio; Treat that the amino acid of coupling and the mol ratio of HOBt are 1:1.
The cracking agent used for volumn concentration be the DCM solution of the TFA of 0.5 ~ 1%, volumn concentration is the DCM solution of the TFE of 20% or TFE and AcOH and the DCM mixture according to volume ratio 1:2:7.Preferred volume percentage composition is the DCM solution of 0.5 ~ 1%TFA.
The tripeptide fragment sequence of the full guard of the middle triptorelin of step (b) and H-Gly-NH
2the condensing agent of condensation is HBTU and HOBt and the combination of the DIEA combination of 1:1:2 in molar ratio or HBTU and HOAt and the DIEA combination of 1:1:2 in molar ratio or DIC and the HOBt combination of 1:1 in molar ratio or EDC and the HOBt combination of 1:1 in molar ratio or PyBOP and HOBt and DIEA 1:1:2 in molar ratio, the combination of preferred HBTU and HOBt and DIEA 1:1:2 in molar ratio; The solvent of condensation reaction is any one or a few the combination in THF, DMF, DCM, NMP and DMSO, preferred DMF.Treat that the carboxyl terminal of coupling and the mol ratio of HOBt are 1:1.
In step (c), the lysate that the cracking of full guard triptorelin uses is TFA and H
2o is the mixing solutions of 95:5 or TFA and EDT and TIS and PhOH and H by volume
2o is the mixing solutions of 80:5:5:5:5 or TFA and EDT and TIS and H by volume
2the mixing solutions of O 92.5:2.5:2.5:2.5 by volume.Preferred TFA and EDT and TIS and H
2the mixing solutions of O 92.5:2.5:2.5:2.5 by volume.
In step (d), purifying is that RPLC purifying changes salt, and namely chromatographic column is C18 post; Moving phase is aqueous acetic acid and acetonitrile solution.
Beneficial effect: hinge structure of the present invention has the following advantages:
1, the present invention utilizes the acid sensitive resin of high loads amount for starting raw material, first adopt the high purity peptide fragment of the selected structure of Solid phase peptide synthesis technology synthesis of standard, liquid phase synthesis difficult sequences, adopt liquid phase coupling technology to make peptide fragment condensation again, thus obtain the target peptide of high purity (>98.5%), high yield (>40%).
2, when synthesizing the polypeptide that there is " difficult sequences ", often fragment condensation is adopted.To compare the technique of continuous solid phase and full liquid phase synthesis triptorelin, the present invention:
A.2-chloro-trityl chloride resin repeated using method is easy, cheap; Each fragment can use the solid phase carrier of high capacity value (>0.8mmol/g resin), and throughput increases;
B. avoiding amino acid and reagent throwing amount in the peptide fragment synthesis of difficult sequences is 1.5-2 times amount, far below Solid phase peptide synthesis routine 3-4 doubly even 5 times, 6 times excessive, save Material Cost;
C. compare full liquid phase synthesis, solid liquid phase binding fragment condensation technology gets the two advantage, has process for solid phase synthesis low material that is easy and liquid phase synthesis concurrently and drops into.
3, adopt the shorter amino acid whose side chain protected peptide fragment sequences purity of super acid responsive type resins synthesis very high, need not purify with chromatographic technique, only need to carry out precipitating, grinding and can use; The coupling of fragment liquid phase, its impurity is mainly the fragment of non-coupling, instead of lacks one or several amino acid whose defect peptide; much easier in final high-efficient liquid phase chromatogram purification; thus number of times is prepared in minimizing, reduces the preparation cost of triptorelin, be conducive to realizing mass-producing, industrialization is produced.
Accompanying drawing explanation
Fig. 1 is the pure peptide analysis color atlas of triptorelin prepared by the present invention;
Fig. 2 is the pure peptide mass spectrum of triptorelin prepared by the present invention.
Embodiment
According to following embodiment, the present invention may be better understood.But those skilled in the art will readily understand, the content described by embodiment only for illustration of the present invention, and should can not limit the present invention described in detail in claims yet.
In the present invention, the peptide fragment aminoacid sequence of the object peptide of synthesis involved by triptorelin and intermediate is in Table l.In the present invention, peptide fragment array mode is in table 2.The implication of the material abbreviation used is in table 3.
The corresponding encoding amino acid sequence of table 1 triptorelin
| Peptide sequence number | Aminoacid sequence | Corresponding encoded amino acid |
| Triptorelin | H-Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH 2 | 1-10 |
| First peptide fragment sequences | H-Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-OH | 1-7 |
| Second peptide fragment sequences | H-Arg-Pro-Gly-NH 2 | 8-10 |
| Tripeptide fragment sequence | H-Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-OH | 1-9 |
Table 2 combined method
Herein, " substitution value " refers to the quantity of the resin-carried material of unit vol, and unit is " mmol/g ".
Table 3 material abbreviation used in the present invention implication
| English abbreviation | Full name |
| Fmoc | 9-fluorenylmethyloxycarbonyl |
| 2-CTC Resin | The chloro-trityl chloride resin of 2- |
| RP-HPLC | RPLC |
| DMF | DMF |
| NMP | N-Methyl pyrrolidone |
| DMSO | Dimethyl sulfoxide (DMSO) |
| DCM | Methylene dichloride |
| THF | Tetrahydrofuran (THF) |
| DIEA | DIPEA |
| NMM | N-methylmorpholine |
| HOBt | 1-hydroxy benzo triazole |
| HOAt | 1-hydroxyl-7-azo benzotriazole |
| PyBOP | Phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl |
| EDC | 1-ethyl-(3-dimethylaminopropyl) carbodiimide |
| HBTU | Benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate |
| DIC | N, N-DIC |
| TFA | Trifluoroacetic acid |
| EDT | 1，2-ethandithiol |
| TIS | Tri isopropyl silane |
| Boc- | Tertbutyloxycarbonyl |
| -Pbf | 2,3-dihydro-2,2,4,6,7-pentamethyl-cumarone-5-alkylsulfonyl |
| -tBu | The tertiary butyl |
| -Trt | Trityl |
Embodiment 1:
1. resin-made is standby
The chloro-trityl resin of preparation Fmoc-Leu-2-: chloro-for 2-trityl chloride resin (5g, substitution value 0.84mmol/g resin, 1eq) is added Peptide systhesis device, with 60mL DCM washing resin.Drain solvent, add the 30mL DCM solution of Fmoc-Leu-OH (1.3eq) and DIEA (2.5eq).This mixture of argon shield mechanical stirring 1 hour.Add chromatogram methyl alcohol 10mL (2ml/g resin) to carry out closing for 30 minutes to the active part on resin.Drain solvent, wash with 3 × 50mL DMF, 3 × 50mL DCM, 3 × 50mL MeOH, vacuum-drying, to constant weight, obtains the chloro-trityl resin of 5.82g Fmoc-Leu-2-.Utilize ultraviolet spectrophotometry to measure Fmoc amount in piperidines deprotection liquid, the capacity value of resin is 0.52mmol/g.
2. fragment preparation
The preparation of 2.1 peptide fragment AA (1-7)-OH:
The chloro-trityl resin of 5g Fmoc-Leu-2-is added in reactive polypeptide room.Add 60mL DCM and stir swellable resins, drain.By 2 × 50mL, 20% piperidines/DMF solution, 5,15 minutes process resin respectively, remove Fmoc.With resin described in 50mLDMF Xian Di 4 times, remove Fmoc by product (dibenzofulvene and its piperidine adduct) and remaining piperidines, ninhydrin reaction mensuration.
Subsequent amino-acid Fmoc-D-Trp (Boc)-OH simultaneously in activation sequences, to react at its C-terminal.The amino acid (2eq) protected by Fmoc-, HOBt (2eq) and DIEA (4eq) are at room temperature dissolved in 25mL DMF.Under argon shield, this solution ice bath is cooled to 0 DEG C, then adds HBTU (2eq), stir dissolving in 5 minutes.The amino acid solution of activation is joined in the resin drained, wash with 5mL DCM.Reactant described in mechanical stirring 1 hour.By qualitative ninhydrin reaction monitoring condensation performance.After the described condensation reaction of judgement completes, then dry adsorbent, with 3 × 50mLDMF washing resin.
Successively with amino acid Tyr (tBu), Ser (tBu), Trp (Boc), His (Trt), each 2eq of Pyr of Fmoc-protection, this operating process is repeated to the follow-up monomer of described peptide fragment.In the end after a coupled reaction, wash with 3 × 50mL DMF, 3 × 50mL DCM, 3 × 50mL MeOH, vacuum-drying, to constant weight, obtains 8.78g resin-bonded peptide.
With 100mL l%TFA/DCM process about 1 hour, 2 × 50mL 0.5%TFA/DCM is then used respectively to wash 5 minutes, from peptide described in resin cracking.Cracking section is collected on pyridine (with TFA volume ratio 1:1).Merge cracking washings, reduced under vacuum is to about 10mL volume, and then with 10mL DMF reconstruct, continuation is simultaneously concentrated is about 10mL to remove remaining DCM to final volume.Add 100mL water precipitation product.This slurry of stirred at ambient temperature 30 minutes.Solid described in collected by vacuum filtration, with about 100mL water washing.Product described in vacuum-drying, obtains 3.84g purity 97%AA (1-7)-OH, productive rate 95%.
The structure of Segment A A (the 1-7)-OH of above-mentioned preparation is as follows:
Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp(Boc)-Leu-OH
Molecular formula: C
88h
105n
11o
15, molecular weight: 1555.778.
2.2 peptide fragment AA (8-10)-NH
2preparation:
110mg H-Gly-NH is added in round-bottomed flask
2hCl (1mmol), 110 μ L NMM (1mmol), 371mgFmoc-Pro-OH (1.1mmol) and 149mg HOBt (1.1mmol).By described dissolution of solid in 20mL THF, then under argon shield, be cooled to 0 DEG C.139mg DIC (1.1mmol) is added in the solution of cooling.0 DEG C of stirred reaction mixture 30 minutes, then rise to ambient temperature overnight.Reaction mixture filters, filtrate concentrates, oily matter acetic acid ethyl dissolution, respectively with saturated sodium bicarbonate aqueous solution, 5% aqueous citric acid solution, saturated sodium-chloride water solution washing, organic layer anhydrous sodium sulfate drying, filters, filtrate rotary evaporation, oily matter MTBE grinds, and obtains 386mgFmoc-Pro-Gly-NH
2, yield 98%.
385mg Fmoc-Pro-Gly-NH is added in round-bottomed flask
2(0.98mmol), slough Fmoc group with 10mL 50% diethylamine/DCM solution, room temperature magnetic stirrer over night, aftertreatment obtains 166mg H-Pro-Gly-NH
2, yield 99%.
714mg Fmoc-Arg (Pbf)-OH (1.1mmol), 165mg H-Pro-Gly-NH is added in round-bottomed flask
2(0.96mmol) with 149mg HOBt (1.1mmol).By described dissolution of solid in 20mL THF, then under argon shield, be cooled to 0 DEG C.139mg DIC (1.1mmol) is added in the solution of cooling.0 DEG C of stirred reaction mixture 30 minutes, then rise to ambient temperature overnight.Reaction mixture filters, filtrate concentrates, oily matter acetic acid ethyl dissolution, respectively with saturated sodium bicarbonate aqueous solution, 5% aqueous citric acid solution, saturated sodium-chloride water solution washing, organic layer anhydrous sodium sulfate drying, filters, filtrate rotary evaporation, oily matter MTBE grinds, and obtains 740mg Fmoc-Arg (Pbf)-Pro-Gly-NH
2, yield 96%.
738mg Fmoc-Arg (Pbf)-Pro-Gly-NH is added in round-bottomed flask
2(0.92mmol), slough Fmoc group, room temperature magnetic stirrer over night with 10mL 50% diethylamine/DCM solution, aftertreatment obtains 522mgH-Arg (Pbf)-Pro-Gly-NH
2, yield 98%.
AA (the 8-10)-NH of preparation
2structure as follows:
H-Arg(Pbf)-Pro-Gly-NH
2
Molecular formula: C
26h
41n
7o
6s, molecular weight: 579.28.
3. fragment condensation process
AA (1-10)-NH is prepared by liquid phase condensations fragment
2
1.556g AA (1-7)-OH (1mmol), 522mg AA (8-10)-NH is added in round-bottomed flask
2(0.9mmol) with 135mg HOBt (1mmol).By described dissolution of solid in 20mL DMF, add 330 μ L DIEA (2mmol), then under argon shield, be cooled to 0 DEG C.379mg HBTU (1mmol) is added in the solution of cooling.0 DEG C of stirred reaction mixture 30 minutes, then rise to room temperature, then stir 1 hour.Add 60mL water precipitation of peptides from described solution.Collected by vacuum filtration solid, with 2 × 50mL water washing, dry acquisition 2.01g crude product AA (1-10)-NH
2.At room temperature grind described solid 3 hours, collected by vacuum filtration with 100mL MTBE, dry acquisition 1.85g AA (1-10)-NH
2, yield 97%.
Reaction process TLC controls, TLC condition: chloroform/methanol/TFE=80:6:6; UV, iodine detects;
AA(1-7)-OH，Rf：0.45；
AA(8-10)-NH
2，Rf：0.28；
AA(1-10)-NH
2，Rf：0.33。
AA (the 1-10)-NH of preparation
2structure as follows:
Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp(Boc)-Leu-Arg(Pbf)-Pro-Gly-NH
2
Molecular formula: C
114h
144n
18o
20s, molecular weight: 2117.05.
4. the cracking of triptorelin and purifying
4.1 by removing side chain protected AA (1-10)-NH
2prepare the thick peptide of triptorelin
Add trifluoroacetic acid/water/tri isopropyl silane/1，2-ethandithiol (92.5:2.5:2.5:2.5) solution 50mL in round-bottomed flask, and be cooled to 0 DEG C.1.12g AA (1-10)-NH is added in this cooling solution
2.Stir described slurry until described dissolution of solid (about 5 minutes) at 0 DEG C, then rise to room temperature, stir 3 hours.This solution is added 0 DEG C of ether 70mL and precipitate described peptide.With 3000rpm centrifugal rotation slurry 5 minutes, from described solid decant ether.By described solid settling flux in 50mL ether, with 3000rpm centrifugal rotation 5 minutes, decant ether.Repeat once, then by dissolution of solid in containing 1% acetic acid 1:1 water/acetonitrile 30mL in, at room temperature preserve 24 hours.By freezing for this solution, then lyophilize obtains the thick peptide of 680mg triptorelin, productive rate 98%.
The thick peptide of 4.2HPLC purifying triptorelin
The thick peptide of 45mg triptorelin produces total length triptorelin sterling 21.1mg through preparation HPLC purifying, productive rate 47%.
HPLC purification condition: chromatographic column: Waters C18250 × 19,5u, 130A; Flow velocity: 8mL/min; Detect: UV, 220nm; Moving phase: A.80% acetonitrile/water; B.0.25% acetic acid/water; Method: 20%-45%A, 10min; 45-60%A, 30min.
The pure peptide color atlas of triptorelin is shown in Fig. 1, and pure peptide mass spectrum is shown in Fig. 2.
The structure of triptorelin is as follows:
Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH
2
Molecular formula: C
64h
82n
18o
13, molecular weight: 1310.63.
Embodiment 2:
1. resin-made is standby
The chloro-trityl resin of preparation Fmoc-Pro-2-: chloro-for 2-trityl chloride resin (5g, substitution value 0.84mmol/g resin, 1eq) is added Peptide systhesis device, with 60mL DCM washing resin.Drain solvent, add the 30mL DCM solution of Fmoc-Pro-OH (1.3eq) and DIEA (2.5eq).This mixture of argon shield mechanical stirring 1 hour.Add chromatogram methyl alcohol 10mL (2ml/g resin) to carry out closing for 30 minutes to the active part on resin.Drain solvent, wash with 3 × 50mL DMF, 3 × 50mL DCM, 3 × 50mL MeOH, vacuum-drying, to constant weight, obtains the chloro-trityl resin of 5.69g Fmoc-Pro-2-.Utilize ultraviolet spectrophotometry to measure Fmoc amount in piperidines deprotection liquid, the capacity value of resin is 0.46mmol/g.
2. fragment preparation
The preparation of peptide fragment AA (1-9)-OH:
The chloro-trityl resin of 5g Fmoc-Pro-2-is added in reactive polypeptide room.Add 60mL DCM and stir swellable resins, drain.By 2 × 50mL, 20% piperidines/DMF solution, 5,15 minutes process resin respectively, remove Fmoc.With resin described in 50mLDMF Xian Di 4 times, remove Fmoc by product (dibenzofulvene and its piperidine adduct) and remaining piperidines, ninhydrin reaction mensuration.
Subsequent amino-acid Fmoc-Arg (Pbf)-OH simultaneously in activation sequences, to react at its C-terminal.The amino acid (2eq) protected by Fmoc-, HOBt (2eq) and DIEA (4eq) are at room temperature dissolved in 25mL DMF.Under argon shield, this solution ice bath is cooled to 0 DEG C, then adds HBTU (2eq), stir dissolving in 5 minutes.The amino acid solution of activation is joined in the resin drained, wash with 5mL DCM.Reactant described in mechanical stirring 1 hour.By qualitative ninhydrin reaction monitoring condensation performance.After the described condensation reaction of judgement completes, then dry adsorbent, with 3 × 50mL DMF washing resin.
Successively with Fmoc-protection amino acid Leu, D-Trp (Boc), Tyr (tBu), Ser (tBu), Trp (Boc), His (Trt), each 2eq of Pyr, this operating process is repeated to the follow-up monomer of described peptide fragment.In the end after a coupled reaction, wash with 3 × 50mLDMF, 3 × 50mL DCM, 3 × 50mL MeOH, vacuum-drying, to constant weight, obtains 9.38g resin-bonded peptide.
With 100mL l%TFA/DCM process about 1 hour, 2 × 50mL 0.5%TFA/DCM is then used respectively to wash 5 minutes, from peptide described in resin cracking.Cracking section is collected on pyridine (with TFA volume ratio 1:1).Merge cracking washings, reduced under vacuum is to about 10mL volume, and then with 10mL DMSO reconstruct, continuation is simultaneously concentrated is about 10mL to remove remaining DCM to final volume.Add 100mL water precipitation product.This slurry of stirred at ambient temperature 30 minutes.Solid described in collected by vacuum filtration, with about 100mL water washing.Product described in vacuum-drying, obtains 4.41g purity 96%AA (1-9)-OH, productive rate 93%.
The structure of Segment A A (the 1-9)-OH of above-mentioned preparation is as follows:
Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp(Boc)-Leu-Arg(Pbf)-Pro-OH
Molecular formula: C
112h
140n
16o
20s, molecular weight: 2061.015.
3. fragment condensation process
AA (1-10)-NH is prepared by liquid phase condensations fragment
2
1.11g AA (1-9)-OH (0.54mmol), 297mg H-Gly-NH is added in round-bottomed flask
2hCl (2.7mmol) and 81mg HOBt (0.6mmol).By described dissolution of solid in 20mL DMF, add 645 μ L DIEA (3.9mmol), then under argon shield, be cooled to 0 DEG C.227mg HBTU (0.6mmol) is added in the solution of cooling.0 DEG C of stirred reaction mixture 1 hour, then rise to room temperature, then stir 1 hour.Add 60mL water precipitation of peptides from described solution.Collected by vacuum filtration solid, with 2 × 50mL water washing, dry acquisition 1.15g crude product AA (1-10)-NH
2.At room temperature grind described solid 3 hours, collected by vacuum filtration with 100mL MTBE, dry acquisition 1.12gAA (1-10)-NH
2, yield 98%.
Reaction process TLC controls, TLC condition: chloroform/methanol/TFE=80:6:6; UV, iodine detects;
AA(1-9)-OH，Rf：0.38；
AA(1-10)-NH
2，Rf：0.33。
AA (the 1-10)-NH of preparation
2structure as follows:
Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp(Boc)-Leu-Arg(Pbf)-Pro-Gly-NH
2
Molecular formula: C
114h
144n
18o
20s, molecular weight: 2117.05.
4. the cracking of triptorelin and purifying are with embodiment 1.
Claims (10)
1. fragment condensation prepares a method for triptorelin, it is characterized in that, solid phase prepares the first peptide fragment sequences of the full guard of triptorelin; Liquid phase is prepared the second peptide fragment sequences of the full guard of triptorelin and is removed its aminoterminal protecting group; In liquid phase, be stripped of the second peptide fragment sequences and the first peptide fragment sequences condensation of the full guard of aminoterminal protecting group, obtain the triptorelin of full guard; The Side chain protective group sloughing the triptorelin of full guard obtains the thick peptide of triptorelin, and high performance liquid phase purifying changes salt and obtains triptorelin acetate;
Wherein,
The first described peptide fragment sequences is 1st ~ 7 amino acids in triptorelin sequence,
The second described peptide fragment sequences is 8th ~ 10 amino acids in triptorelin sequence.
2. fragment condensation according to claim 1 prepares the method for triptorelin, it is characterized in that, it comprises the following steps:
(1) solid phase prepares the first peptide fragment sequences resin of the full guard of triptorelin,
Described full guard first peptide fragment sequences is:
Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp(Boc)-Leu-OH，
By full guard first peptide fragment sequences from cracking solid phase carrier;
(2) the second peptide fragment sequences of the full guard of liquid phase synthesis triptorelin,
Second peptide fragment sequences of described full guard is:
Fmoc-Arg(Pbf)-Pro-Gly-NH
2；
Remove the aminoterminal protecting group of the second peptide fragment sequences of full guard;
(3) the first peptide fragment sequences condensation of the full guard that the second peptide fragment sequences being stripped of the full guard of aminoterminal protecting group step (2) obtained in liquid phase and step (1) obtain obtains full guard triptorelin;
(4) Side chain protective group is sloughed in the cracking of full guard triptorelin and obtain triptorelin crude product;
(5) the purified salt that turns of triptorelin crude product obtains triptorelin acetate.
3. fragment condensation according to claim 2 prepares the method for triptorelin, it is characterized in that, in step (1), the first peptide fragment sequences resin of full guard is coupled at successively on solid phase carrier by amino acid to obtain; Wherein, solid phase carrier used is acid sensitive resin;
Solid phase is prepared in the process of triptorelin full guard first peptide fragment sequences resin, and the amino deprotecting regent used is the DMF solution of the DBU of 1% for volumn concentration is the DMF solution of the piperidines of 20% or volumn concentration;
Coupling agent used is the combination of HBTU and HOBt and the combination of DIEA or the combination of DIC and HOBt or PyBOP and HOBt and DIEA;
The cracking agent used for volumn concentration be the DCM solution of the TFA of 0.5 ~ 1%, volumn concentration is the DCM solution of the TFE of 20% or TFE and AcOH and the DCM mixture according to volume ratio 1:2:7.
4. fragment condensation according to claim 2 prepares the method for triptorelin, it is characterized in that, in step (2), the method for the second peptide fragment sequences of liquid phase synthesis full guard is, first Fmoc-Pro-OH and H-Gly-NH
2be obtained by reacting Fmoc-Pro-Gly-NH
2, slough Fmoc protection after and Fmoc-Arg (Pbf)-OH be obtained by reacting Fmoc-Arg (Pbf)-Pro-Gly-NH
2;
The described aminoterminal protecting group removing the second peptide fragment sequences of full guard namely sloughs Fmoc-Arg (Pbf)-Pro-Gly-NH
2fmoc protection, obtain H-Arg (Pbf)-Pro-Gly-NH
2;
Amino deprotecting regent is the DMF solution of the piperidines of volumn concentration 16%, or volumn concentration is the DMF solution of the DBU of 1%, or volumn concentration is the DCM solution of the diethylamine of 50%; The combination that the condensing agent that condensation reaction uses is HBTU and HOBt and the combination of DIEA or the combination of the combination of HBTU and HOAt and DIEA or the combination of DIC and HOBt or EDC and HOBt or PyBOP and HOBt and DIEA; The combination that the reaction solvent that condensation reaction uses is any one or a few in THF, DMF, DCM, NMP and DMSO.
5. fragment condensation according to claim 2 prepares the method for triptorelin, it is characterized in that, in step (3), first peptide fragment sequences condensation of the full guard that the second peptide fragment sequences being stripped of the full guard of aminoterminal protecting group step (2) obtained and step (1) obtain obtains in full guard triptorelin process, the combination that the condensing agent of use is HBTU and HOBt and the combination of DIEA or the combination of the combination of HBTU and HOAt and DIEA or the combination of EDC and HOBt or DIC and HOBt or PyBOP and HOBt and DIEA; The solvent of condensation reaction is any one or a few the combination in THF, DMF, DCM, NMP and DMSO.
6. fragment condensation prepares a method for triptorelin, it is characterized in that, solid phase prepares the tripeptide fragment sequence resin of the full guard of triptorelin, then the tripeptide fragment sequence of full guard is got off from cracking resin, with H-Gly-NH in liquid phase
2condensation obtains the triptorelin of full guard, and then slough side chain protected and obtain triptorelin crude product, purifying turns salt and obtains triptorelin acetate;
Wherein, described tripeptide fragment sequence is the 1-9 amino acids in triptorelin sequence.
7. fragment condensation according to claim 6 prepares the method for triptorelin, it is characterized in that, it comprises the following steps:
A () solid phase prepares the tripeptide fragment sequence resin of the full guard of triptorelin,
The tripeptide fragment sequence of the full guard of described triptorelin is:
Pyr-His(Trt)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Trp(Boc)-Leu-Arg(Pbf)-Pro-OH，
By the tripeptide fragment sequence of full guard from cracking solid phase carrier;
Full guard tripeptide fragment sequence and H-Gly-NH in (b) liquid phase
2condensation obtains the triptorelin of full guard;
C the triptorelin cracking of full guard is sloughed Side chain protective group and is obtained triptorelin crude product by ();
D the purified salt that turns of () triptorelin crude product obtains triptorelin acetate.
8. fragment condensation according to claim 7 prepares the method for triptorelin, it is characterized in that, in step (a), the tripeptide fragment sequence resin of the full guard of triptorelin is coupled at successively on solid phase carrier by amino acid to obtain peptide resin; Wherein, solid phase carrier used is acid sensitive resin;
Solid phase is prepared in the process of tripeptide fragment sequence resin of the full guard of triptorelin, and the amino deprotecting regent used is the DMF solution of the DBU of 1% for volumn concentration is the DMF solution of the piperidines of 20% or volumn concentration;
Coupling agent used is the combination of HBTU and HOBt and the combination of DIEA or the combination of DIC and HOBt or PyBOP and HOBt and DIEA;
The cracking agent used for volumn concentration be the DCM solution of the TFA of 0.5 ~ 1%, volumn concentration is the DCM solution of the TFE of 20% or TFE and AcOH and the DCM mixture according to volume ratio 1:2:7.
9. fragment condensation according to claim 7 prepares the method for triptorelin, it is characterized in that, the tripeptide fragment sequence of the full guard of the middle triptorelin of step (b) and H-Gly-NH
2the condensing agent of condensation is the combination of HBTU and HOBt and the combination of DIEA or the combination of the combination of HBTU and HOAt and DIEA or the combination of DIC and HOBt or EDC and HOBt or PyBOP and HOBt and DIEA; The solvent of condensation reaction is any one or a few the combination in THF, DMF, DCM, NMP and DMSO.
10. fragment condensation according to claim 7 prepares the method for triptorelin, it is characterized in that, in step (c), the lysate that the cracking of full guard triptorelin uses is TFA and H
2o is the mixing solutions of 95:5 or TFA and EDT and TIS and PhOH and H by volume
2o is the mixing solutions of 80:5:5:5:5 or TFA and EDT and TIS and H by volume
2the mixing solutions of O 92.5:2.5:2.5:2.5 by volume.
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